In solid-phase peptide sequencing, a polypeptide is immobilized on a solid support, and a series of chemical reactions are carried out sequentially to release and identify amino acid residues from the C- or N-terminal end of the polypeptide (Coull).
The most widely used method for N-terminal sequencing involves reacting the N-terminal amino group of the polypeptide with phenyl isothiocyanate (PITC), in a process known as Edman degradation (Edman). The reaction of PITC with the terminal amino group adds a phenylthiourea group, which cyclizes and cleaves, forming a free anilinothiozolanone (ATZ) of the N-terminal amino acid, and a shortened peptide. The ATZ-derivative of the N-terminal amino acid is separated, converted to the corresponding phenylthiohydantoin (PTH), and identified by high performance liquid chromatography (HPLC). Sequencing is then carried out by successively converting the next-in N-terminal residue to a free amino acid PTH, and identifying each successively released amino acid. The method is generally reliable for sequences up to about 20-40 amino acid residues and is readily performed with automated instrumentation.
At the present time, most C-terminal sequencing involve the formation of a C-terminal thiohydantoin (TH) or thiohydantoin-like derivative. In one approach, the C-terminal carboxyl group of a polypeptide is activated using acetic anhydride in the presence of an isothiocyanate (ITC) salt or acid to form a C-terminal thiohydantoin via a C-terminal ITC intermediate (Stark). The C-terminal thiohydantoin can be cleaved from the polypeptide, producing a shortened peptide and the thiohydantoin derivative of the C-terminal amino acid residue. This derivative can be separated and identified, e.g., by HPLC.
Two general approaches have been used for immobilizing polypeptides on a solid support for sequence analysis. In one approach, the polypeptide is immobilized by covalent attachment to the support via reactive groups on the support. For N-terminal sequencing, the C-terminal carboxylic acid group can be reacted with an activating reagent, such as carbonyldiimidazole or carbodiimide, for subsequent coupling to support-bound amino groups (Laursen). Covalent attachment via side chain groups of internal residues is also possible (Findlay and Geisow, 1989).
For N-terminal sequencing, DITC-activated glass is commonly used, where support-bound isothiocyanate groups react with polypeptide amino groups (i.e., lysyl .epsilon.-amino groups) to form stable thiourea linkages with the polypeptide (Bridgen). The .alpha.-amino group of the polypeptide can also react with the support, but such linkages (with .alpha.-amino groups) can be cleaved using trifluoroacetic acid (TFA) (Allen, 1981).
Covalent attachment using activated supports offers the advantage of essentially permanent immobilization of the polypeptide on the support, thereby minimizing sample wash-out. However, activated supports tend to be susceptible to inactivation by water. Thus, polypeptide samples must be dissolved in special, non-aqueous solvents (e.g., acetonitrile) prior to immobilization on the support. In addition, the efficiencies of immobilization are often inconsistent because the activating groups deteriorate over time.
In a second approach, the polypeptide is immobilized noncovalently. In gas-liquid solid-phase sequencing, for example, the sample is typically entrained in a membrane-type support by ionic and hydrogen-bonding interactions with the polypeptide. Glass fiber supports have proven useful for this application, although other support-types can be used. For example, proteins resolved by SDS-polyacrylamide gel electrophoresis can be electroblotted directly onto polyvinylidene difluoride (PVDF) membranes, and the part of the membrane containing the protein of interest can be loaded directly into an automated sequencer (Matsudaira, 1987).
Although supports for non-covalent binding avoid the problems of activating group stability, such supports usually fail to retain small polypeptides. The efficiency and longevity of immobilization can often be improved by adding to the support a polycationic carrier, such as POLYBREEN, which forms a matrix on the surface of the support to enhance retention of the polypeptide. One drawback, however, is that such matrices usually require precycling, i.e., treatment of the matrix-coated support with several sequencing cycles prior to sample loading, to remove contaminants from the matrix which would otherwise interfere with amino acid identification during sequence analysis. Thus, use of such matrices can slow the sequencing protocol.
Ideally, an activated support for covalent immobilization of polypeptides should react readily with polypeptides. The support should be compatible with aqueous polypeptide samples, and should be storable for extended periods of time without losing binding efficiency.